1. Field of the Invention
This invention pertains generally to devices and methods for sifting components of heterogeneous mixtures, and more particularly to devices and methods for separating and quantifying biological particulates with different physiological states with an optoelectronic tweezers and dielectrophoresis (OET-DEP) separation scheme.
2. Description of Related Art
The efficient separation of biological particulates such as stem cells, embryos or bacteria from isolates without damaging the particulates is an important step in a variety of diagnostic and treatment methods.
There are a variety of applications where it is desirable to sort heterogeneous populations of cells. One example is the sorting of adult stem cell populations based on cell viability. It may be desirable to exclude dead or minimally viable stem cells from living, more hearty/viable cells, for use in human and/or animal stem cell medical therapeutics, reproductive-assistance interventions, genetic-screening of gametes, and in preparation for cell “banking” (cell storage by cryogenic or other modalities).
Another example is the sorting of non-motile viable sperm from non-motile non-viable sperm. In a subset of infertile human patients or animals, where nearly all sperm are non-motile, it is desirable to sort viable from dead sperm, for use with “in-vitro” insemination procedures. The limitation encountered with present technologies and modalities is that it is not possible to use the same sperm that have been sorted into “non-motile/live” and “non-motile/dead” groups, because either the sorting modality is lethal to the sperm, or, because it poses unacceptable risk of genetic damage to the sorted sperm so as to preclude using the same sperm, if found to be “viable”, for fertilization.
A further useful sorting would be the sorting of cells based on chromosome number, such as for screening to exclude gametes with an abnormal number of chromosomes or sorting of cells based on chromosome structural damage. Specific examples include normal chromosome damage due to cell aging or acquired cell damage from various cell processing techniques, radiation, chemotherapy, and iatrogenic tissue injury.
Sorting of cells based on genetic mutations, despite otherwise “intact” chromosome structure is also desirable. Examples include genetic mutations that code for aberrant proteins and/or protein structures that give rise to syndromes/disease states of a living organism. One illustration is the Cystic Fibrosis (CF) mutation, which causes CF, a common hereditary disease caused by mutations of the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated anion channel, with clinical manifestations of progressive lung disease, pancreatic insufficiency, and infertility in both sexes. CF is one of the most significant life-shortening autosomal recessive disorders found in Caucasians worldwide.
Sorting of cells based on the type of chromosome(s) contained within the cell may also be useful. For example, gamete “sex-sorting” for use in assisted-reproduction techniques, where the pre-selection the sex of the offspring is desirable or where it is essential that the sex of the offspring is ensured to be either male or female, in order to avoid propagation of sex-specific genetic disorders/diseases.
Accordingly, the separation of live cells from dead cells, cancer cells from normal cells or gram-positive from gram-negative bacteria can be important for diagnostic and therapeutic treatments. These applications require that cells not only be sorted efficiently and into a fine spectrum of groups, but also that the cells can be retrieved for use at the end of the sorting procedure.
Existing approaches to cell sorting are often labor intensive and create damage risks to the cells being sorted from mechanical forces, or chemical exposures. For example, physical or genetic damage may occur through mechanical sorting (i.e. the use of filter systems) or flow Cytometry assays or differential uptake of chemicals (“cell viability” assays).
In contrast to other techniques used to sort particles, dielectrophoresis (DEP) does not require that the cell be motile; the presence or knowledge of cell surface antigens; or the use of external materials, such as antibodies or chemicals to aid visualization. Dielectrophoresis refers to the motion of neutral particles as a result of the application of an external non-uniform electric field. Unlike linear electrophoresis, DEP does not require the object to have a net charge.
A non-uniform electric field interacts with an induced dipole in the particle and the particle can experience a dielectrophoretic force toward a region of high field intensity (positive dielectrophoresis) or toward a region of low field intensity (negative dielectrophoresis). In other words, the DEP forces attract some particles and repel other particles. The phenomenon can be observed with either AC or DC electric fields because dielectrophoretic forces do not depend on the polarity of the electric field. Therefore, the motion of the particle is not a result of its polarity but of the magnitude of the electric field.
Previous devices use a DEP field that has been generated by a “fixed electrode” embedded within a micro-chamber filled with a minimally conductive solution and particles to be studied. Each electrode is independent and connected to a source of alternating current, and thus generates a DEP field at its location. This “fixed electrode” design does allow one to use DEP to “trap” a particle so that it can be visually examined, or, to examine which cells travel toward the electrode. However, the “fixed electrode” design is extremely limited. Fixed electrodes also require costly microfabrication, produce bubbles and electrolysis products that can harm device operation, and can damage cells with their strong field gradients.
For example, to “study” even a small group of cells, each cell must be manually isolated and delivered to the fixed DEP field. This reliance on delivering each cell to the source of the DEP field is very labor intensive, and creates the risk that the cells being studied will be damaged during such extensive manipulation.
Another limitation of the “fixed electrode” design is that because the source of DEP is fixed in space, it cannot be moved. Therefore, it is very difficult to measure the attraction each particle for the DEP field. The cell must be placed a distance away from the DEP electrode, to see how fast it moves towards the electrode. Such measurements are compromised by the fact that the DEP field diminishes with distance. Therefore, the DEP field that a particle experiences a given distance away from the electrode is, by definition, different from what experiences closer or further away. The effect of distance from the electrode, on the DEP field strength the particle experiences can be accounted for by mathematical models, but this is tedious, and introduces the possibility of systematic and random measurement errors.
Living cells can be described as “dielectric” objects because they can be electrically polarized in an electric field due to their inherent electric gradients. Living cells maintain electrical gradients across their semi-permeable cell membranes. The dielectric potential of any cell has been shown to depend on its physiologic status; composition (e.g., charged membrane structures, cytoplasm contents, organelles, and charged protein and DNA); morphology; and phenotype, in addition to the frequency of the applied electrical field. Therefore, the same cell type in different physiological states (which differ with respect to the latter factors define the dielectric potential) will possess distinctly different dielectric potentials, which in turn can be utilized for separation.
Accordingly, there is a need for Micro-fluidic cell-sorting chip designs to facilitate sorting of heterogeneous populations of living and dead cells as well as select by viability, cell size, magnitude of cell membrane dipole, and features of the cell's chromosome content, such as chromosome number, degree of chromosome damage, chromosome type (gamete sex-sorting), and genetic aberrations of known and unknown diseases. There is also a need for sorting devices that are high through-put and sort cells into a spectrum rather than just two groups. There is a further need for devices that can retrieve the sorted cells.
The present invention satisfies the need for greater sorting throughput, greater sorting specificity, and the efficient retrieval of the sorted product and is generally an improvement over the art.